A functional immune system has to maintain a delicate balance between effective elimination of foreign pathogens and minimizing collateral damage to the host. Regulatory T cells (Tregs), a subset of T lymphocytes, function as suppressor of the immune system and play a critical role in maintaining immune homeostasis. Defects in Treg function lead to autoimmune diseases and immunopathology in humans and mice. One of the milestones in Treg biology field was the identification of transcription factor Foxp3 as an essential regulator of Treg development and function. The goal of this project is to elucidate the molecular mechanisms that control Foxp3 expression, which will determine how regulatory T cells are generated and maintained. By comparing Foxp3 genomic sequences of different species, Dr. Zheng and colleagues identified three intronic regions that are evolutionarily conserved, which they named CNS1, 2, and 3 (Conserved Non- coding Sequence). They hypothesized that these CNS regions could play an important role in regulating Foxp3 expression, and proceeded to knockout individual Foxp3 CNS regions in mice. Each of the three CNS knockout mice showed different defects in their Treg population, suggesting they play different roles in control of Foxp3 expression. CNS3 is required for thymic development of Tregs, whereas CNS1 is involved in TGF-beta dependent peripheral conversion of Tregs. Most interestingly, CNS2 plays a role in maintaining Foxp3 expression and stabilizing Treg lineage. In the current proposal, Dr. Zheng's group will follow up on the initial characterization of the Foxp3 CNS knockout mice to perform a detailed study on how the CNS regions control regulatory T cell homeostasis at both cellular and molecular levels. First, they will use more physiologically relevant mouse models to investigate the role of Foxp3 CNS2 in maintenance of natural Treg lineage stability and perform TCR repertoire study to examine the flux of Treg subsets in the absence of CNS2 (Aim 1). Second, they will explore the role of Foxp3 CNS regions in generation and maintenance of antigen-specific Tregs induced in vivo by anti-DEC205 chimeric antibody (Aim 2). Lastly, they plan to use a modified chromatin immunoprecipitation assay to identify proteins bound to CNS regions that regulate Foxp3 expression (Aim 3). The study proposed here will provide more insight on mechanisms controlling Treg homeostasis, and potentially generate new avenues to manipulate Tregs to attenuate or augment the amplitude of immune response for treatment of autoimmune diseases and cancer.